JP2007500363A - Liquid permeable composition in dry reagent apparatus - Google Patents

Abstract

  A multilayer device (16) for detecting an analyte in a fluid sample, the first absorbent layer (12) for receiving and absorbing a portion of the sample from the first layer (10) And a liquid permeable layer (14) that is disposed between the first absorbent layer (10) and the second absorbent layer and acts as an adhesive to hold both layers together. Multi-layer device.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US patent application Ser. No. 10 / 459,825, filed on Jun. 12, 2003, which is incorporated herein by reference in its entirety. This application claims priority to IB03 / 00055, which claims priority from US Provisional Application No. 60 / 348,253, filed Jan. 15, 2002.

BACKGROUND OF THE INVENTION Diagnostic dry reagent analyzers are common products used in the clinical setting of urine analysis and blood tests, particularly glucose monitoring. Results are obtained instrumentally or visually as thresholds and quantitative outputs. A dry reagent analyzer typically includes an absorbent pad containing a dispersed reagent system that reacts with an analyte (a component to be detected) in a fluid sample applied to the device to produce a detectable response. These reagents contain indicator dyes, metals, enzymes, polymers, antibodies and various other chemicals that are dry deposited on the carrier. Commonly used carriers are papers, membranes or polymers with a variety of sample absorption and transport properties.

  Some reagent strips use only one reagent zone to contain all the chemicals necessary to generate a color response to the analyte. In some cases, up to five competitive and synchronous chemical reactions occur in one reagent layer. The Hemastix® reagent strip (Bayer), a method for detecting blood in urine, is an example of a number of chemical reactions that occur with one reagent. The analyte detection reaction is based on the peroxidase-like activity of hemoglobin that catalyzes the oxidation of the indicator 3,3 ', 5,5'-tetramethyl-benzidine by diisopropylbenzene dihydroperoxide. At the same pad, a second reaction takes place to eliminate ascorbic acid interference based on the catalytic activity of the ferric-HETDA complex that catalyzes the oxidation of ascorbic acid by diisopropylbenzene dihydroperoxide.

  Typical chemical reactions that occur in dry reagent strips can be classified as dye-binding reactions, enzymatic reactions, immunological reactions, and redox catalyzed reactions. Dye binding to an analyte such as albumin results in a color change at the micromolar level. The indicator dye can be covalently bound to the analyte (diazonium compound that binds to bilirubin) or can be tightly associated with the analyte (sodium sensitive indicator). Enzymatic reactions can be used to detect enzymes at the micromolar level through reaction with color forming substrates. Enzymatic reactions can also be used to detect molecules such as glucose by producing a colored end product through reaction with the enzyme. Particle-labeled antibodies are the primary reagents that provide a detectable response for chromatographic-based immunological strips. Redox catalysis involves the use of a metal chelate compound to oxidize or reduce an indicator in the presence of a specific analyte such as hemoglobin and can detect molecules down to the nanomolar level. Certain of these devices include an enzymatic reaction with the analyte in the presence of peroxidase and hydroperoxide to produce a detectable discoloration of the redox dye, usually filter paper as an absorbent pad Based on the use of.

  Dry reagent devices often include multiple reagent layers to measure a single analyte. This variant allows the chemical reagent system to be placed in a separate reagent layer and allows for reaction separation steps such as chromatography and filtration. Immunochromatographic strips are constructed so that chemical reactions occur in separate layers of reagents. The CLINITEST® hCG strip test (Bayer) for human chorionic gonadotropin is an example of a dry reagent strip test with four reagent layers. The first layer at the tip of the strip is the layer for sample application and overlaps the next reagent layer to provide transfer of the patient sample (urine) to the first reagent zone. And the processed sample moves in the 3rd layer in which the reactant for coloring is fixed. This movement is driven by a fourth pad that absorbs excess sample. The chromatographic reaction occurs in a third layer, typically a nitrocellulose membrane, called the test or capture zone. In the first and second layers, the analyte specific antibody reacts with the analyte in the sample and is transferred to the nitrocellulose membrane by chromatography. The antibody is bound to colored latex particles as a label. If the sample contains the analyte, the analyte reacts with the labeled antibody. In the capture zone, the second antibody is immobilized on the band and captures the particles when the analyte is present. A colored test line is formed. A second reagent band is also immobilized in the capture zone, allowing the control line to react with the particles and form a color. If the test system is working correctly, the color of the control line will always form, even if no hCG is present in the patient sample.

  Whole blood glucose strips often use multiple reagents to capture intact red blood cells that interfere with the color generation layer. An example is GLUCOMETER Encore® (Bayer), which uses a capture layer placed directly above the color generation layer. The color is read from the bottom of the strip through a transparent window. Other designs allow the sample to move to a color generation layer that is separate from the acquisition layer, and the color is read from the top of the strip. Whole blood test strips often use a plastic cassette to hold the reaction layer in place. Numerous reagent layers have also been applied to reagent systems used with film slides, such as the Ektachem analyzer (Vitros) developed by Eastman Kodak (1980). The slides were capable of enhancing color using multiple separation layers, development and color forming layers.

  These dry reagent devices are inexpensive and easy to use, but have some limitations. For example, immunoassays require the separation of components to function, which is often accomplished by protein binding. The movement of reagents and analytes often presents problems and gives inaccurate results. The connection between the layers is critical for obtaining accurate results, and in many cases the fluid transfer between these layers is difficult to control. In a dry reagent format such as that described by Greenquist US Pat. No. 4,806,311 the analyte is combined with the labeling reagent and then passed through a detection zone where the analyte is analyzed by the amount of labeling reagent. The amount of objects is measured. Unreacted labeling reagent is immobilized by the immobilized analyte in the reagent zone. The labeled reagent-analyte passed through the detection zone cannot move in the reverse direction by being immobilized in the detection zone.

  The assembly and manufacture of multilayer devices is not completely successful. For example, in EP 0226465A2 and US Pat. No. 3,992,159, a film is used to separate reagent layers. However, these devices require close control of the film pore size, shape and thickness. As a result of such a design, the reagent cannot be placed on the filter paper. The reason is that such filter paper does not have a well-defined film pore structure or uniform surface required for uniform thickness. However, filter paper is desirable for multilayer devices because it is well suited for the use of many reagents due to its inert nature and high water absorption. Therefore, filter paper has been used with nylon mesh covers. Such devices rely on surface contact between reagent layers, which mix reagents into one layer on the surface. The present invention avoids this result and maintains the reagents in their intended positions.

  There are numerous examples of chemicals that are incompatible with dry reagent systems. For example, bases in leukocyte reagents cause premature hydrolysis of protease substrates. Iron in the occult blood reagent causes premature oxidation of the redox dye indicator to a colored form, which is also a result of the presence of iodate in the glucose reagent. In the case of copper-based tests for creatinine, copper can oxidize redox indicators such as tetramethylbenzidine to colored forms even in the absence of creatinine. Urine occult blood testing can be distorted by the presence of ascorbate in the urine sample, which acts as a reducing agent and produces false negative results, and the urine protein test is a buffer in the urine sample being tested May be inaccurate due to the presence of Dry test devices for measuring white blood cells in urine may be subject to interference by proteins in urine samples, and whole blood tests such as blood glucose and blood CKMB suffer from interference caused by red blood cells. In one embodiment, the present invention provides adhesive properties, comprising two layers, at least one of which contains a reagent for detection of an analyte, an aqueous polymer dispersion and a water-soluble polymer. Casting to form a layer having and separating with a test fluid permeable composition comprising a dried blend provides a means to alleviate these problems.

  Previous methods for dealing with these problems involved dividing the reagent into separate layers. However, there are problems associated with the use of separate stacked layer structures. For example, the top layer should allow the sample to pass to the layer below it while continuing to separate certain interfering chemicals and / or biochemicals. For example, metals such as copper or iron should be separated from redox indicators and bases should be separated from protease substrates. Oxidants such as iodate and reactants such as ascorbate must be separated from redox indicators such as tetramethylbenzidine.

  These problems are factors that act to remove interfering substances as they pass through the first layer of the device, through the permeable composition of the present invention and into the second layer of the device. It is effectively handled by derivatizing the permeable composition. This multilayer format requires a permeable adhesive to hold the reagent layer together.

  However, in the prior art, the contact between the layers was insufficient for the reactants to pass from one layer to the adjacent layer when desired or moved from one layer to another when not desired. It was like that.

  There are a variety of diffusible adhesive compositions that can be used to bond the two layers in an integrated multilayer reagent device. Verbeck, US Pat. No. 3,993,451, uses an adhesive to secure reagent-containing particles to a substrate layer. The particles may be covered with a porous layer, and components contained in the sample may pass through the layer to reach the reagent-containing particles. In the device devised by Verbeck, the adhesive is not used as a layer separating the reagent layer from the detection layer. Furthermore, the solid particles form a separate detection unit that does not rely on movement of the reaction product with the analyte to the adjacent layer for detection.

  Japanese Patent Laid-Open No. 05-18959 discloses the use of a hydrophobic polymer that does not swell in water as an adhesive for fixing the reagent layer, and Japanese Patent Laid-Open No. 05-26875 is an adhesive for fixing the reagent layer. Discloses the use of a porous layer comprising a fluorine-containing polymer. The polymers used in these Japanese systems are hydrophobic and thus prevent rapid movement of sample fluid through the layers. For rapid testing, the sample fluid should pass through the layers of the device in less than 1 second. Water soluble adhesives allow for rapid movement of the sample fluid, but will cause the layers to separate as the adhesive begins to dissolve.

  EP 0226465 A2 describes a multilayer analyzer in which several porous sheets are joined by an adhesive arranged to form an opening through which liquid can pass. The adhesive itself cannot pass liquids, and therefore is provided with openings instead. As a result, not all available surfaces are useful and the contact between layers is not uniform.

  The Greenquist No. 311 patent mentioned above discloses a multilayer device for medical testing. The concept is valuable, but in practice, multilayer devices are not as satisfactory as desired. The layer must perform its intended function without interfering with the function of the adjacent layer. At the same time, the sample fluid must pass quickly through the layers so that the results can be determined quickly. Thus, the layers must act independently without limiting sample fluid movement. We solve these problems as described below for multilayer devices and microfluidic devices.

  U.S. Pat. No. 4,824,640 discloses a transparent layer that is useful for containing analytical reagents, consisting of a water-soluble or water-swellable component and an essentially insoluble film-forming component. In US Pat. No. 6,187,268, a similar layer is used as an overcoat over the dry reagent layer.

  The above types of dry reagent strips are not the only test method used near the patient. Microfluidic devices have been developed that have advantages over multilayer dry reagent strips. The general principles of certain microfluidic devices that are of interest to the inventors are found in US patent application Ser. No. 10 / 082,415. Microfluidic devices are designed to receive small liquid samples, such as blood and urine, and then process them through chambers interconnected by capillary passages. The chamber can contain reagents that react with the components in the sample as required for the intended analysis. The challenges inherent in multilayer test strips can be avoided. The necessary reactions can occur sequentially as the sample or part of the sample is transferred from one chamber to another, typically by capillary or centrifugal forces. Thus, as described in further detail below, the present invention can be applied to microfluidic devices in addition to multilayer dry test strips.

SUMMARY OF THE INVENTION The present invention includes a liquid sample comprising a liquid permeable layer capable of acting as an adhesive disposed between an absorbent layer or a non-absorbable layer, wherein at least one of the three layers contains a reagent. And a method and apparatus for detecting an analyte within. The liquid permeable adhesive layer is permeable to the components of the fluid sample and is cast and dried to form a layer that can act as an adhesive and an aqueous polymer dispersion and a water soluble polymer. Contains a blend of In one embodiment, the liquid permeable adhesive layer is disposed between at least the first absorbent layer and the second absorbent layer in the reagent reservoir of the microfluidic, strip or cassette device. At least one of the layers contains a reagent system for detection of the analyte. In another embodiment, a liquid permeable adhesive layer is disposed between two non-absorbable layers in a reagent reservoir of a microfluidic chip or cassette device. In yet another embodiment, the liquid permeable layer is disposed between a plurality of alternating absorbent or non-absorbable layers in the reagent reservoir of the microfluidic chip or cassette device. In all embodiments, the absorbent layer and the adhesive layer may or may not contain reagents.

  The water dispersible polymer can be an anionic or cationic polyurethane dispersion, preferably an anionic polyurethane, in combination with a water soluble polymer, preferably polyethylene oxide, polyvinyl pyrrolidone or polyvinyl alcohol.

  The liquid permeable composition used in the present invention can be used to construct several types of multilayer devices that include a liquid permeable composition between two absorbent or non-absorbent layers. The liquid permeable composition with adhesive holds the separate layers together. One layer is a plastic support base or cover, such as a strip handle, cassette top or bottom, or microfluidic device cover or base so that a person using the device can avoid direct contact with the sample fluid. be able to. Because the adhesive composition is permeable, reagents and fluid sample components can flow from one layer to another.

  The multi-layer device can be manufactured such that when a fluid sample is placed on the first absorbent layer, it deploys on the surface of the layer without interacting with the components of the sample. Alternatively, the first absorbent layer may react with the interference component of the sample so that the measured component (analyte) passes through the liquid permeable layer and reaches the second absorbent layer. it can. Alternatively, the first absorbent layer can be reacted with the analyte and measured at a given location, or the reaction product can pass through the liquid permeable layer to the second absorbent layer where it is detected. Can also be done. The second absorbent layer can absorb and retain the components of the fluid sample that have passed through the adhesive layer, and can also be the analyte or reaction product of the analyte received from the first absorbent layer. Reactive reagents can also be included. The liquid permeable layer can be manufactured to prevent the passage of sample components due to physical separation. For example, it may act to concentrate the analyte by preventing other components from reaching the second absorbent layer while passing through the analyte. Alternatively, the liquid permeable layer can contain reagents that react chemically with specific components of the sample components. In one embodiment, the liquid permeable layer passes certain components of the sample, leaving a more concentrated analyte in the first absorbent layer.

In a preferred embodiment, the permeable adhesive layer can contain exchange resins and ascorbate scavengers to eliminate buffer and ascorbate interference from the sample. Cation exchange resins include oxidizing anions such as those having bromic acid, iodic acid, periodic acid and chromate ions or polysulfonic acids, polycarboxylic acids or polyphosphonic acids together with transition metal oxidants such as iron, cobalt or copper There is something to contain. The permeable adhesive layer also contains a protein binding polymer to separate the interfering protein or antibody from the sample and a filler to adjust the opacity or reflection behavior of the reagent device, eg TiO ₂ or BaSO ₄ be able to. Suitable protein binding polymers include, for example, positively charged polymers such as polyamines and polyamides and negatively charged polymers such as polysulfonic acids, polycarboxylic acids and polyphosphonic acids. These polymers can be incorporated into the permeable layer by incorporating them into the adhesive formulation and coating the reagent layer.

  In a microfluidic device, the liquid permeable composition can be placed in the reservoir of the device to pass only the liquid sample or its components. In some embodiments, the multilayer device described above can also be adapted to function in a sample reservoir of a microfluidic device. In other embodiments, the liquid permeable composition can be placed at the inlet or outlet of the sample reservoir or can fill the reservoir. In such applications, the liquid permeable composition can also contain additives for reacting with the components in the sample to prepare the sample for further reaction, as in the multilayer strip described above.

Detailed Description of the Invention Layer Materials Three common materials are used in biological dry fluid samples, particularly in laminated dry reagent strips or microfluidic chips for analysis of blood and urine. These three materials can be placed in many designs depending on the requirements of the analysis being performed. These materials are generally referred to as absorbent materials, non-absorbable materials and permeable materials.

  Biological samples are generally aqueous, so that the absorbent layer has the ability to absorb aqueous materials. Thus, the absorbent layer can generally be classified as hydrophilic. Useful materials for the absorbent layer include cellulose, nitrocellulose, nylon, glass, porous polyethylene and polyester. When a biological sample is placed in the absorbent layer, the liquid sample moves through the layer, limited by the amount of sample and the reaction of the reagent placed in the absorbent layer with the sample. If only one reagent is applied to the absorbent layer, it is important to distribute the sample uniformly so that a consistent response is obtained. If more than one reagent is applied to the absorbent layer, the sample must be contacted with each reagent simultaneously or sequentially if necessary. Part or all of the sample moves to the side opposite to the side where the sample is located, thereby providing the components of the original liquid sample and mobile reaction products on the opposite side.

  The non-absorbable layer, of course, does not absorb biological samples and is often hydrophobic, but for example non-porous plastic films may be hydrophilic and non-absorbable. Usually, the sample placed on the surface of the non-absorbing layer moves to the extent that the difference in interfacial energy between the sample and the non-absorbing layer allows. Often the surface is hydrophobic so that the liquid sample can be confined to a predetermined area on the layer. For example, the reagent can be applied to areas on the disposed non-absorbent layer such that a portion of the sample does not move between such areas.

  The permeable layer has the ability to transfer liquid from one layer to another, but in a different manner than the absorbent layer described above. The permeable layer is not porous and its composition can be adjusted to allow the liquid to move through it at different rates depending on the analysis being performed. In many applications, a permeable layer is in intimate contact with an adjacent absorbing or non-absorbing layer, thus effectively transferring a liquid sample or a portion thereof to another adjacent surface across the permeable layer. be able to. Such close contact with adjacent layers can provide adhesion between the layers, which places the reagent-containing layer in the desired location when the multilayer test strip is assembled or when a permeable material is used in the microfluidic chip. It is advantageous to fix to.

Multilayer Device In one simple example shown in FIG. 1, a multilayer device for detecting an analyte (ie, a substance to be detected) in a fluid sample is a first absorbent layer 10 for receiving a fluid sample. Receiving a portion of the sample from the first absorbent layer and being disposed between the second absorbent layer 12 and the two absorbent layers for absorbing and holding the absorbent layers together It includes a liquid permeable layer 14 that acts as an adhesive. The liquid permeable layer not only binds the absorbent layer together, but can react with the components of the fluid sample to prevent their passage or physically block the passage of the components of the fluid sample. Three layers are attached to the handle 16. As will be apparent to those skilled in the art, additional absorbent and liquid permeable layers may be added as needed to perform a particular analysis.

  The first absorbent layer has several possible functions. It is also possible to simply absorb the fluid sample and deploy it on the surface of the liquid permeable layer and the adhesive layer. Alternatively, the analyte can react with the interference component of the sample and pass the analyte through the liquid permeable layer to the second absorbent layer. As another alternative, after the first absorbent layer has reacted with the analyte, the analyte is measured in place, or the reaction product is passed to the second absorbent stratification for detection. Pass through the liquid permeable layer.

  The second absorbent layer also has some potential functions. A portion of the sample that has passed through the liquid permeable layer can be absorbed, thereby concentrating the analyte in the first absorbent layer. Alternatively, after receiving a portion of the sample containing the analyte, it can react with the analyte to provide a product to be measured. As another alternative, the second absorbent layer may receive the reaction product produced in the first absorbent layer and concentrated by passage through the liquid permeable layer.

  The liquid permeable layer concentrates the analyte by allowing the analyte to pass through the second absorbent layer and not allowing other components to pass through the second absorbent layer, or by allowing interference components to pass through. By doing so, the fluid sample can be physically separated. In other applications, the liquid permeable layer may react with specific components of the sample, thereby trapping those components in the liquid permeable layer. Alternatively, it may contain additives that can react with certain components, thereby preventing the passage of those components through the liquid permeable layer.

  Those skilled in the art will appreciate that this broad description of the functionality of the multilayer device of the present invention also applies to many specific alternative applications, some of which are discussed below, but other applications not mentioned are also described in this document. It will be understood that this is a potentially useful analytical method without missing the broad description of the invention.

  Furthermore, the target analysis technique may be implemented by appropriately including a non-absorbable layer in the multilayer device. FIG. 2 shows one possible design. The first non-absorbent layer 20 is used to send a fluid flow to and from the second non-absorbent layer 24 to the liquid permeable layer 22, and conversely, the liquid permeable layers are layers 20 and 24. Provide liquid contact between

Microfluidic device In another embodiment of the present invention, a microfluidic device uses a liquid permeable composition with a dry reagent. The microfluidic analyzer can also be called a “chip”. These are generally small and flat, typically about 1 to 2 inches square (25 to 50 mm square) or similar sized disks (eg, radius 25 to 120 mm). The amount of sample supplied to the microfluidic device is small. For example, it contains only about 0.3-1.5 μL. The reservoir that receives the sample liquid is relatively wide and shallow so that the sample can be easily viewed and measured with a suitable instrument. The capillary passages interconnecting the reservoirs range in width from 10 to 500 μm, preferably from 20 to 100 μm, and their shape depends on the method used to form the passages. The minimum allowable depth of the passage can be determined by the nature of the sample. The depth should be at least 5 μm, but should be at least 20 μm when whole blood is the sample. If capillary segments are used to define a predetermined amount of sample, the capillary may be larger than the passage between the reagent reservoirs.

  There are several ways in which capillaries and sample reservoirs can be formed, such as injection molding, laser ablation, diamond grinding or embossing, but it is preferable to use injection molding to reduce the cost of the chip. In general, the base portion of the chip is cut to form a sample reservoir and a desired network of capillaries, and then the top portion is attached on the base to complete the chip.

  The chip is intended to be disposed of after a single use. Thus, it is made of a material that is as inexpensive as possible and at the same time compatible with the reagents and sample to be analyzed. In most cases, the chip is made of plastic, such as polycarbonate, polystyrene, polyacrylate or polyurethane, but can alternatively be made of silicate, glass, wax or metal.

  For a given passage, the interaction between the liquid and the passage surface may or may not have a significant effect on the movement of the liquid. When the surface to volume ratio of the passage is large, that is, the cross-sectional area is small, the interaction between the liquid and the walls of the passage becomes very significant. This is especially true when the capillary force prevails over the liquid sample and wall interface energy for passages with a nominal diameter of less than about 200 μm. When the wall is wet with liquid, the liquid moves in the passage without receiving external force. Conversely, if the wall is not wet with liquid, the liquid will try to escape from the passage. Utilizing these general trends, liquid can be moved in a passage or stopped at a junction with another passage having a different cross-sectional area. If the liquid is stationary, it can be moved by applying a force, such as a centrifugal force. Alternatively, other means, including air pressure, vacuum, electroosmosis, absorbents, further capillary action, etc., that can induce the required pressure differential at the junction between passages with different cross-sectional areas or interfacial energy May be used. If the passage is very small, the force due to the capillary action allows the liquid to be moved only by the force due to the capillary action without the need for an external force, except for a short period of time when the capillary stopper must be overcome. However, smaller passages are inherently more likely to be blocked by particles in biological samples or reagents. As a result, the interfacial energy of the passage walls is adjusted according to the requirements for use with the sample fluid being tested, such as blood, urine and the like. This allows for more flexible analyzer design.

  The capillary passage can be adjusted to either hydrophobic or hydrophilic as defined with respect to the contact angle formed on the solid surface by the liquid sample or reagent. Usually, a surface is considered hydrophilic if the contact angle is less than 90 ° and hydrophobic if the contact angle is greater than 90 °. Preferably, plasma polymerization is performed on the passage surface. The analytical device of the present invention can also be manufactured by other methods used to control the interfacial energy of the capillary wall, such as coating, grafting or corona treatment of hydrophilic or hydrophobic materials. It is preferable to adjust the interfacial energy of the capillary wall, ie, the degree of hydrophilicity or hydrophobicity, for use with the intended sample fluid. For example, to prevent deposits on the walls of the hydrophobic passage or to ensure that no liquid remains in the passage.

  Movement of the liquid through the capillary channel can be blocked by a capillary stopper that prevents the liquid from flowing through the capillary channel, as the name implies. If the capillary passage is hydrophilic and facilitates liquid flow, a hydrophobic capillary stopper, i.e. a smaller passage with hydrophobic walls, can be used. Because the combination of small size and non-wetting walls creates a surface tension that opposes liquid penetration, the liquid cannot pass through the hydrophobic stopper. Alternatively, if the capillary is hydrophobic, no stopper is required between the sample reservoir and the capillary. The liquid in the sample reservoir overcomes the surface tension countered by the liquid and is prevented from entering the capillary until sufficient force, such as centrifugal force, is applied to pass the liquid through the hydrophobic passage. Centrifugal force is sufficient to start the liquid flow. Once the walls of the hydrophobic passage are in full contact with the liquid, the counter force is reduced because the presence of the liquid lowers the energy barrier associated with the hydrophobic surface. As a result, the liquid no longer requires centrifugal force to flow. Although not necessary, in some cases it may be advantageous to continue to apply centrifugal force as the liquid flows through the capillary passage to facilitate rapid analysis.

  When the capillary passage is hydrophilic, the sample fluid (assumed to be aqueous) naturally flows through the capillary without requiring additional force. If a capillary stopper is required, one alternative is to use a narrow hydrophobic section that can act as a stopper as described above. Even if the capillary is hydrophilic, a hydrophilic stopper can be used. One such stopper is wider than the capillary, so the surface tension of the liquid creates a lower force that promotes the flow of the liquid. If the difference in width between the capillary and the wide stopper is sufficient, the liquid stops at the entrance to the capillary stopper. The liquid eventually penetrates along the hydrophilic wall of the stopper, but with proper shape design, this movement can be sufficiently delayed so that the stopper is effective even if the wall is hydrophilic. all right. Alternatively, the hydrophilic stopper can result in a sudden narrowing of the passage so that liquid does not flow through the narrow passage until an appropriate force, such as centrifugal force, is applied.

  Microfluidic devices can be designed in a number of ways to perform the kind of analysis currently being performed using the multilayer strips described above. Alternatively, because the sample reservoir is divided in the microfluidic device, it is possible to minimize undesirable interactions between components in the liquid sample or reagents used to perform the analysis. . In some cases, the reservoir contains one reagent for performing one step of the analytical method. However, in the present invention, the liquid permeable composition can be used in a variety of ways, some of which are similar to those in multilayer applications. For example, as shown in FIG. 3, the liquid permeable composition 32 may be disposed between two dry reagents 30 and 34 in one reservoir. A liquid sample runs up the ramp 36 and contacts the three-layer reagent, while air in the reagent reservoir is purged from the vent 38. Alternatively, the liquid permeable composition can be placed at the inlet or outlet of the sample reservoir to perform a filtration function, i.e., remove some components of the sample before the sample reaches the reagent. Further, if desired, the entire sample reservoir may be filled with a liquid permeable composition. Other possible applications include the application of filling the capillary with a liquid permeable composition and the application of additional layers of absorbent and / or non-absorbent materials for the adhesive to adhere to the inside of the device. . For example, the reservoir can contain 10 very thin layers with a permeable adhesive between each layer. The sample stream can be directed to a predetermined area of each layer by the arrangement of absorbent and non-absorbable materials. Adhesion is useful to ensure that the composition remains in place and to avoid movement that may cause the sample to bypass it.

Liquid Permeable Adhesive Composition The basic elements of a liquid permeable composition useful in the present invention include an aqueous polymer dispersion and a water soluble polymer. The permeability of the composition can be adjusted by changing the ratio of the polymer dispersion to the water soluble component. Usually this ratio is 50: 1 to 1: 1 on a weight basis, with a ratio of 10: 1 to 5: 1 being excessive in film-forming polymer dispersion. Increasing the water dispersible polymer increases the permeability of the membrane, which is desirable when higher flow rates are desired. Conversely, if greater contact, and thus more mixing of reagents, is desired, increasing the concentration of the water-soluble polymer decreases the membrane permeability. In a dry diagnostic reagent testing device, components present in a fluid sample are infiltrated into a permeable layer that ties together the reagent layers of the device. In microfluidic devices, the liquid permeable composition has a similar function but may not always come into contact with the dry reagent.

  Polyurethane dispersions are preferred for use as dispersible polymers because of their adhesion, flexibility and diverse structures. Provided is a linear polyurethane in which a reaction between a diisocyanate and an equivalent amount of a difunctional alcohol is simple. These products are not suitable for use in the production of coatings, paints and elastomers. However, first a simple glycol is reacted with a dicarboxylic acid in a polycondensation reaction to form long chain polyester-diols, followed by reaction of these products, generally having an average molecular weight of 300-2000, with diisocyanates. As a result, a high molecular weight polyester urethane is formed. Polyurethane dispersions have been commercially important since 1972. Polyurethane ionomers are structurally suitable for the preparation of aqueous two-phase systems. These polymers with hydrophilic ionic sites between predominantly hydrophobic chain segments are self-dispersing and, under favorable conditions, are not affected by shear forces, even in the absence of dispersants. To form a stable dispersion. In order to obtain a preferred anionic polyurethane for use in the present invention, such as Bayhdrol DLN, diols having carboxylic acid or sulfonate groups are introduced and then the acid groups are neutralized with, for example, tertiary amines. Because these compounds are water soluble, the sulfonate group is usually constructed via diaminoalkane sulfonate. The resulting polyurethane resin builds ionic groups that provide mechanical and chemical stability and good film-forming adhesion.

  Cationic polyurethane dispersions such as Praestol E 150 from Stockhausen Chemical can also be used to form liquid permeable compositions. One method of preparing the cationic polyurethane is by a reaction of dibromide and diamine. If one of these components contains a long chain polyester segment, an ionomer is obtained. Alternatively, a polyammonium polyurethane can also be prepared by first preparing a tertiary nitrogen-containing polyurethane and then quaternizing nitrogen atoms in the second step. Starting from polyether-based NCO prepolymers, segmented quaternary polyurethanes are obtained.

  The most important property of polyurethane ionomers is to provide a two-component colloidal system in which a discontinuous polyurethane layer is dispersed in a continuous aqueous phase by forming a naturally stable dispersion in water under certain conditions. Is the ability to The diameter of the dispersed polyurethane particles can vary from about 10 to 5000 nm. Particularly suitable are polyurethane dispersions which are ionic and whose ionic groups are sulfonate, carboxylate or ammonium groups.

  Also suitable for use in the present invention are other film-forming polymer dispersions such as those formed by polyvinyl or polyacrylic compounds such as polyvinyl acetate or polyacrylate, vinyl copolymers, polystyrene sulfonic acids, polyamides And mixtures thereof. Combining the polymer dispersion with the water-soluble polymer forms a matrix that produces a swellable networked web. The denser the web, the smaller the pores and the slower the flow of test fluid in the matrix.

  As the water-soluble polymer, known polymers such as polyacrylamide, polyacrylic acid, cellulose ethers, polyethyleneimine, polyvinyl alcohol, copolymers of vinyl alcohol and vinyl acetate, gelatin, agarose, alginate and polyvinylpyrrolidone are suitable. This second polymer component is sometimes called a swelling component because of its property of swelling by absorbing water. Polyethylene oxide, polyvinyl pyrrolidone and polyvinyl alcohol are preferred. These polymers are water soluble and can vary greatly in molecular weight as long as they are miscible with the aqueous polymer dispersion. Polyethylene oxide having a molecular weight of 300,000 to 900,000 g / mol and polyvinylpyrrolidone having a molecular weight of 30,000 to 60,000 g / mol are particularly suitable. The molecular weight of the water-soluble polymer is not critical as long as it is miscible with the polymer dispersion and allows the incorporation of specific reagents such as buffers, indicators, enzymes and antibodies for testing. The finished film should be swellable so that it is permeable to the test fluid.

  The polymer is dispersed / dissolved in a solvent (preferably aqueous) for application to a dry reagent device or microfluidic chip by use of a dispenser, as in the examples below. In a preferred aqueous casting solution, the aqueous polymer dispersion is mixed with an aqueous solution of the second polymer. For example, a polyvinyl acetate dispersion is mixed with cellulose ethers, a polyurethane dispersion is mixed with polyvinyl alcohol, a polyurethane dispersion is mixed with gelatin, or a polyurethane dispersion is mixed with polyvinylpyrrolidone. Typically, a surfactant is added to the formulation to increase its spreadability and a thickener, such as silica gel, is added to thicken the formulation to a consistency that facilitates deployment on the surface. The formulation is then applied to the surface of the dry reagent device or microfluidic chip with a Myer rod applicator or wiping film spreader and dried to remove the solvent. Typical dry thicknesses for permeable membranes range from 1 to 100 mils (0.0254 to 2.54 mm).

Use of Liquid Permeable Compositions Protein interference in the testing of urine leukocytes is mitigated by the protein adhering to the liquid permeable composition and not passing through the reagent. Buffer interference in urine protein testing is reduced by adhering to the liquid permeable composition (ion pairing) or neutralizing (proton exchange), so that the buffer does not come into contact with the reagent. To a non-interfering form that is compatible with the pH of the reagent. The device can be manufactured to hold two separate reagent layers, one with copper and the other with a redox indicator, so to test urine creatinine due to the presence of incompatible chemicals The instability of these reagents is mitigated by the liquid permeable composition. The copper is kept separate from the redox indicator until it contacts the fluid sample. The sample supplies creatine to bind with copper, which is released from the upper layer and mixed with the redox indicator.

  Ascorbate interference to the urine occult blood test can be mitigated by incorporating into the liquid permeable composition an ascorbate scavenger, such as a metal capable of oxidizing ascorbate bound to the polymer. Polymer bonded metal ascorbate scavengers are described in US Pat. No. 5,079,140. Other oxidizing agents such as iodate and persulfate can be immobilized in the permeable composition to act as an ascorbate scavenger.

  Liquid permeable compositions can be advantageously used with immune formats to provide high sensitivity testing of various analytes. For example, a transparent membrane for use in a multilayer device can be prepared with an immobilized anti-binding labeled antibody contained therein. Typically, the antibody is incorporated into the membrane by attaching it to a larger entity, e.g., latex particles that are incorporated into the polymer blend to form the membrane, before casting the polymer blend onto a reagent device. It is fixed with. Thus, if the binding label on the anti-analyte antibody has a fluorescein structure (eg, fluorescein isothiocyanate (FITC)), anti-FITC is dispersed in the permeable membrane and the anti-analysis labeled with FITC The target antibody can be captured. In addition, peroxidase-labeled anti-analyte antibody is formulated into the membrane and when the test fluid flows through the membrane, the analyte contained therein is bound to the anti-analyte antibody and peroxidase-labeled anti-analysis. Binds to the target antibody to form a sandwich attached to the membrane, thereby preventing peroxidase from reaching the reagent layer containing the peroxide and redox dye and providing a color response. In this embodiment, the response produced by the interaction of the analyte, peroxidase, peroxide and redox dye is inversely proportional to the concentration of the analyte in the fluid sample.

  More generally, non-limiting examples of reagents that can find use in the multilayer or microfluidic devices of the present invention include:

Reagents for reaction with the analyte in the first absorbent layer that receives the fluid sample include enzymes commonly used in clinical trials, such as oxidases, reductases and proteases, and chemistry that allows the analyte to be detached. There are affinity binders such as antibodies, nucleic acids, antigens and proteins, such as those used in binding tests and reactions that are converted to substances.

-Reagents for reaction with interfering components of fluid samples include enzymes for metabolizing interfering substances, reactants for converting interfering substances into non-reactive forms, and binders for capturing interfering substances .

Reagents for reaction with the analyte in the second absorbent layer include indicators that emit signals in response to the analyte and enzymes or reactants for signal amplification.

-Reagents for reaction with the analyte in the second absorbent layer that reacted in the first absorbent layer and passed through the adhesive layer include enzymes used in clinical trials and analytes There are affinity binders used in binding tests and reactions in which a portion of the object is cleaved.

Additives to the liquid permeable composition that can react with the components of the sample include affinity binders or enzymes to remove interfering substances or emit signals.

  The following provide five examples illustrating alternative embodiments of the present invention, but are not intended to limit the present invention to these examples. In one embodiment, the filter paper layer is treated with a reagent solution for the analyte to be detected. Then, the treated filter paper is coated with the adhesive layer of the present invention, the second layer of untreated filter paper is added, reacts with the analyte, and moves through the adhesive layer to become untreated filter paper. It can act to concentrate incoming reagents. In a second embodiment, the adhesive layer includes a substance that prevents interference compounds from passing through the adhesive layer and into the reagent layer. A third example includes an upper layer containing reagents for the analyte. The product of the reaction between the analyte and the reagent passes through the adhesive layer and is detected in the lower layer.

Example I
The diffusible adhesive was prepared as follows.
(1) 75 g of 50 mM monobasic phosphate buffer (Fisher, pH 7.0) and 0.5 g of Pluronic P75 surfactant (BASF) were added to a 250 ml steel beaker. Next, 0.3 g of octanol and then 5.0 g of Aerosil 200 silica gel (DeGussa) were added to the beaker with slow stirring. Agitation speed was increased to about 2000 rpm for several minutes to achieve complete dispersion of the contents of the beaker.

  (2) While stirring was continued for about 15 minutes, 40.25 g of a 40% aqueous solution of Bayhydrol D-762 (Bayer's polyester polyurethane resin) was added, followed by m. w. 0.2 g of 900,000 polyethylene oxide was added.

  (3) The coating solution was stirred for several minutes under a weak vacuum to degas the solution and ready for casting on the reagent layer. The albumin reagent layer was prepared as follows.

  (1) Two solutions are prepared for sequential application to a filter paper base. The composition is shown in the table below.

  (2) Filter paper (Whatman GF / 30 cm) was treated with two solutions in order to impregnate the paper, and then the treated filter paper was dried at 90 ° C. for 15 minutes to prepare an upper layer reagent.

  After the adhesive coating solution is cast on the albumin reagent layer to a thickness of about 250 μm (wet), the adhesive-coated albumin reagent on the filter paper is dried at about 90 ° C. for 5 minutes, and the coating adheres to the albumin reagent layer. The hardened upper surface was exposed.

  A complete format was assembled in which a layer of glass filter paper (Whatman GF / 30 cm) was placed on the side of the adhesive layer opposite the albumin reagent layer. That is, the test apparatus included three layers: an albumin reagent layer, a diffusible adhesive layer adhered to the albumin reagent layer, and a layer of glass filter paper that did not adhere to the cured exposed surface of the diffusible layer. This demonstrated that once the film was fully cured, it did not bond to further layers. The test device was manufactured as described above but compared to an albumin reagent layer that was not coated with a diffusible adhesive layer.

  In the first test, a sample containing 500 mg / L of albumin was applied to an albumin reagent layer without an adhesive coating, and the results were compared to another 500 mg / L sample placed on the glass filter paper of the composite device. . In the latter case, albumin had to pass through the filter paper and the adhesive layer in order to reach the reagent layer where it was detected. In the comparative sample, the reagent layer gave a quick response. The amount of albumin present was measured by reflectometry using a CLINITEK 200 instrument. When no sample was added to the albumin reagent layer, the reflectance was 93.6% at a wavelength of 610 nm at 1 minute from the start of analysis. However, the reflectivity was found to be 12.8% when the sample was added directly to the reagent layer without the adhesive layer. When the sample was applied to glass filter paper and the sample reached the reagent layer by passing through the filter paper and adhesive, the reflectivity was found to be 13.0%. It can be concluded that the filter paper and adhesive do not substantially affect the composition of the sample that passes through them and reaches the reagent layer.

  In the second test, a much lower concentration, 1 mg / L of albumin was used. In this case, the albumin reagent without the adhesive coating showed a reflectivity of 52.4% indicating a lower concentration of albumin in the sample. However, when the sample was placed directly on the albumin reagent layer in the composite device, the reflectivity was measured to be 25.4%, indicating a higher response to the same concentration of albumin. It can be concluded that some of the liquid in the sample passes through the adhesive and enters the filter paper layer, thereby increasing the effective concentration of albumin on the reagent layer.

Example II
In this example, a binding reagent layer was added to the diffusible adhesive layer to remove competing or interfering components, thereby allowing the analyte to reach the detection reagent layer. In the same manner as the adhesive composition described in Example I, a protein-blocked diffusible adhesive composition was prepared as follows.

  (1) 150 g of 0.1 M sodium citrate buffer at pH 5.5 and 1.0 g of Pluronic L64 surfactant were added to a 250 ml steel beaker. While slowly stirring, 0.6 g of octanol was added, 12.0 g of Aerosil 200 was added, and stirring was continued for several minutes at about 2000 rpm to complete the dispersion of the components.

  (2) While continuing stirring, 110 g of a 40% aqueous solution of Bayhydrol DLN was added, and then 0.4 g of PEO 900,000 was added. Mixing was continued for about 15 minutes.

  (3) 100 μL of casein blocking solution was added to each 1 g of the coating solution completed in step (2). The mixture was swirled to produce a uniform coating solution. The adhesive coating solution was cast as a 100 μm layer on the peroxidase reagent layer.

Peroxidase reagent layer
(A) preparing a 10 mg / ml solution of 3,3 ′, 5,5′-tetraethylbenzidine,
(B) Immerse Whatman 3mm filter paper into the solution,
(C) drying the impregnated paper at 40 ° C. for 15 minutes,
(D) The paper dried in step (c) is immersed in a solution of raw material glucose oxidase 1400 U / ml,
(E) The paper impregnated in step (d) was prepared by drying at 40 ° C. for 20 minutes.

  (4) The adhesive-peroxidase reagent layer combination was pressed onto the binding reagent layer without application of heat using a roller laminator with a force of 100 ft / min and 100 psi and dried at 40 ° C. for 30 minutes.

The binding reagent layer is
(A) The following ingredients
Dralon L (Polyacrylonitrile) 17.3g
Ultrason E (polyether polysulfone) 69.1 g
Aerosil 200 (silica) 25.9g
Pluriol P600 (propylene oxide surfactant) 7.78g
A polymer membrane from
(B) Immerse the membrane in a solution of 4 mg / ml anti-FITC (anti-fluorescein isothiocyanate) in 0.1 M sodium citrate (pH 4.5 buffer)
(C) The impregnated filter paper was prepared by drying at 40 ° C. for 30 minutes.

  (5) The combined layers were dried at room temperature for 1-2 hours to complete the preparation of the analyzer.

  In the test, the samples contained both BSA-FITC (bovine serum albumin-anti-fluorescein isothiocyanate) and HRP-FITC (horseradish peroxidase-anti-fluorescein isothiocyanate), the latter competing with BSA-FITC. It was. BSA-FITC separated from HRP-FITC as the sample passed through the adhesive layer. HRP-FITC reached the peroxidase reagent, color development occurred and indicated its presence, and reflectance was measured with a CLINITEK® 50 analyzer. When only HRP-FITC is present, the reflectivity was found to be 62.6%, but when BSA-FITC is present, the reflectivity is 45.7%, when excess FITC is achieved. It was shown that HRP-FITC can permeate the membrane.

  A comparative test was performed in which the binding layer and the peroxidase reagent layer were placed in contact with each other without an intermediate adhesive layer. In this case, no difference was observed between the two samples. That is, there was no separation of samples containing both BSA-FITC and HRP-FITC. Even if excess FITC is present in the tie layer, it can be concluded that it is impossible to separate competing analytes without an adhesive.

Example III
This example illustrates the use of a multilayer device similar to the device of Example II to measure digoxin. Reagents and protein binding layers containing a substrate capable of detecting peroxidase were prepared as described in Example II. These layers were combined with the diffusible adhesive layer as described above to produce a three-layer device. After drying, the combined layers were cut into strips and each strip was covered with a polystyrene strip with a square opening that served as a sample reservoir. Prepare samples containing digoxin 0, 25, 50 and 100 μg / ml and 50 ml of digoxin-BSA-HRP (digoxin-bovine serum albumin-horseradish peroxidase) 50 mg / ml solution and anti-digoxin labeled FITC 50 mg / ml solution did. 45 μl of each sample mixture was added to the sample reservoir on the strip to contact the sample with the protein binding layer. The sample passed through the top and adhesive layers and entered the reagent layer, where a color response was emitted. Measurements performed with a CLINITEK® 50 reflectance spectrometer showed that digoxin reached the reagent layer in proportion to its concentration in the sample, as shown in the table below.

  In comparative tests that did not include the adhesive layer, no difference in response from the reagent layer was seen, indicating that no competition occurred without the adhesive layer.

  Examples IV and V illustrate the use of a multilayer device similar to the device of Example II for measuring glucose using a microfluidic chip as a reagent holder.

Example IV
One example of using a permeable adhesive in a microfluidic device is measuring the glucose content in the blood. The glucose reagent described in Bell US Pat. No. 5,360,595 was prepared on an absorbent layer, for example, a nylon membrane such as Pall Biodyn. A permeable adhesive formulation as described in Example 1 was then coated onto the glucose reagent. The reagent area was placed in a microfluidic reagent reservoir with the permeable adhesive facing up or down. When placed face down, the adhesive bound to the microfluidic base, and when placed face up, the glue bound to the non-absorbent plastic lid covering the chamber. Other layers of absorbent or non-absorbable materials can also be applied as layers.

  The inlet was used to introduce a blood sample containing a concentration of glucose into the reagent chamber. When the whole blood sample reacted with the reagent and developed color, it was read by a spectrometer at 680 nm, corrected for black and white standards.

Example V
The glucose reagent described in Bell US Pat. No. 5,360,595 was prepared by coating the reagent on a plastic absorbent substrate such as PES and PET. When using reagent-coated PET, the reaction with the sample was read using a 500-950 nm reflectometer. The permeable adhesive was coated over the glucose reagent to flow through the permeable adhesive. As long as the sample flow from the inlet to the reagent is not obstructed by the non-absorbing material, the adhesive is bound to the microfluidic base, a non-absorbing plastic lid or other layer of absorbent or non-absorbing material covering the chamber. . The inlet was used to introduce a blood sample containing a concentration of glucose into the reagent chamber. The whole blood sample reacted with the reagent and developed color. Since the plastic film is transparent, a 500-950 nm reflectometer was used to read the reaction with the sample corrected for black and white standards.

Example VI
The effects of film thickness and curing temperature were examined and the results are summarized in the following table. A layer of permeable adhesive having a composition similar to that of Example I was applied to the filter paper layer, and an upper layer of filter paper or PET was applied. The three layers were cured at 90 ° C. or 40 ° C. with and without pressure.

  A permeable adhesive was added over one of the filter paper layers and precured in an oven at 40 ° C. for 20 minutes or 90 ° C. for 5 minutes. Next, another roller paper layer was laminated to the membrane adhesive using a roller laminator at 100 ft / min and using 0 or 40-100 psi force without applying heat. The combined three layers were cured at substantially ambient temperature (30 ° C.) for 2 hours to complete the cure. Case 8 was similar to the method used in Examples II-V.

  The above table reports the state of the three layers after curing using the following criteria:

  Tear seal refers to whether a portion of the outer layer, not the adhesive interlayer, is damaged when the outer layer is pulled apart.

  A bubble refers to the formation of an area where the adhesive is not adhered, as determined by adding water to one of the outer layers and detecting the area where the adhesive is not adhered to the outer layer.

  Permeability was determined by adding water to one of the outer layers and measuring the time taken for the water to reach the other layer.

  The delamination means evaluation of separation between layers when the three layers are cut with a commercially available apparatus, punched out and pressed.

  Double-sided adhesion is associated with tear seal evaluation. If a tear seal is found on one or both of the two outer layers, i.e. the outer layer breaks rather than the adhesive layer, it is stated that double-sided adhesion has not been achieved.

  Examining the results in the table, it can be concluded that the double-sided adhesion is not achieved unless the lamination pressure is applied. When laminating pressure was applied, double sided adhesion occurred when the thickness met the initial application temperature. For example, a 10 mil thick layer was not double-sided when initially cured at 90 ° C, but double-sided when initially cured at 40 ° C. If the film adhesive reaches full dryness, i.e., cures, it will not act as a double-sided adhesive. The use of laminating pressure has unexpectedly eliminated problems with bubbles and delamination and permeability. For example, a thick film such as 100 mil in Case 3 contained bubbles and was not usable because it had poor permeability unless pressurized. Typically, high temperatures and thickness reduced permeability and lower temperatures caused delamination. It was surprising that pressure solved these problems in the 10-100 mil thickness range.

FIG. 3 is a cross-sectional view of a dry reagent device having three layers, an absorbent upper layer, a liquid permeable adhesive layer, and an absorbent lower layer. FIG. 2 is a cross-sectional view of a dry reagent device having three layers: a non-absorbable upper layer, a liquid permeable adhesive layer, and a non-absorbable lower layer. FIG. 3 is a cross-sectional view of a reagent reservoir in a microfluidic chip containing stacked reagents.

Claims (66)

  An apparatus for detecting an analyte in a liquid sample, the apparatus comprising: a liquid permeable layer capable of adhering to transfer a part of the liquid sample, wherein the liquid permeable layer includes the liquid A blend of an aqueous polymer dispersion and a water-soluble polymer, dried to form a permeable layer, wherein the liquid permeable layer is applied as a coating between an absorbent layer or a non-absorbent layer The combined coating and the absorbent and non-absorbent layers are pressed and dried at a temperature such that the liquid-permeable layer retains liquid permeability while adhering to an adjacent or non-absorbent layer ,apparatus.   The apparatus of claim 1, further comprising an additive in the liquid permeable layer that chemically reacts with a component in the liquid sample.   The device of claim 2, wherein the additive in the liquid permeable layer comprises an indicator dye or particles for emitting a detectable response.   The apparatus of claim 2, wherein the additive comprises an exchange resin for removing buffer components and an ascorbate scavenger for eliminating ascorbate interference.   The apparatus of claim 2, wherein the additive comprises particles and a polymer for producing a detectable response or removing an interfering component.   The apparatus of claim 2, wherein the additive comprises a metal and a chelating compound for producing a detectable response or removing an interfering component.   The device of claim 2, wherein the additive comprises an enzyme for producing a detectable response or removing an interfering component.   The device of claim 2, wherein the additive comprises an antibody or other affinity molecule for generating a detectable response or for separating interference components.   The apparatus of claim 2, wherein the additive comprises a filler for adjusting opacity or reflectivity.   The apparatus of claim 2, wherein the additive comprises a surfactant to increase fluid flow.   The device of claim 1, wherein the liquid permeability of the liquid permeable layer is adjusted by changing the ratio of the aqueous polymer dispersion to the water soluble polymer.   The apparatus of claim 11, wherein the aqueous polymer dispersion is a polyurethane dispersion and the water-soluble polymer is at least one member of the group consisting of polyethylene oxide, polyvinyl pyrrolidone and polyvinyl alcohol.   13. The apparatus of claim 12, wherein the ratio of the aqueous polymer dispersion to the water soluble polymer is 50: 1 to 1: 1 on a weight basis.   A method of analyzing a liquid sample by contacting a liquid sample with a dry reagent in a layer that reacts with an analyte in the sample to produce a detectable response, wherein a portion of the liquid sample is in the layer A liquid permeable layer comprising a blend of an aqueous polymer dispersion and a water soluble polymer for transferring between and adhering to the layer, wherein the liquid permeable layer is applied as a liquid coating and is liquid permeable A method wherein the layers are pressed and dried in place between the layers at a temperature such that the layers remain bonded while adhering to the layers.   15. The method of claim 14, wherein the liquid permeable layer concentrates the sample by passing liquid in the sample through the layer.   The method of claim 14, wherein the liquid permeable layer includes an additive that chemically reacts with a component in the liquid sample.   The method of claim 16, wherein the additive comprises an indicator dye or particle for producing a detectable response.   The method of claim 16, wherein the additive comprises an exchange resin to remove buffer components and an ascorbate scavenger to eliminate ascorbate interference.   The method of claim 16, wherein the additive comprises particles and a polymer for producing a detectable response or removing an interfering component.   17. The method of claim 16, wherein the additive comprises a metal and a chelate compound for producing a detectable response or removing an interfering component.   17. The method of claim 16, wherein the additive comprises an enzyme for producing a detectable response or removing an interfering component.   17. The method of claim 16, wherein the additive comprises an antibody or other affinity molecule for generating a detectable response or for separating interference components.   The method of claim 16, wherein the additive comprises a filler for adjusting opacity or reflectivity.   The method of claim 16, wherein the additive comprises a surfactant to increase fluid flow.   15. The method of claim 14, wherein the liquid permeability of the liquid permeable layer is adjusted by changing the ratio of the aqueous polymer dispersion to the water soluble polymer.   26. The method of claim 25, wherein the aqueous polymer dispersion is a polyurethane dispersion and the water soluble polymer is at least one member of the group consisting of polyethylene oxide, polyvinyl pyrrolidone and polyvinyl alcohol.   27. The method of claim 26, wherein the ratio of the aqueous polymer dispersion to the water soluble polymer is 50: 1 to 1: 1 on a weight basis. A multi-layer device for detecting an analyte in a fluid sample,
(A) at least a first absorbent or non-absorbent layer for receiving the fluid sample;
(B) at least a second absorbent or non-absorbable layer for receiving a portion of the sample from the first layer, and (c) a liquid disposed between the at least first and second layers. Including a permeable adhesive layer,
The adhesive is diffusible into a fluid, cast as a liquid coating, and at a temperature such that the adhesive layer retains liquid permeability while adhering to the first and second layers. An apparatus comprising a blend of an aqueous polymer dispersion and a water soluble polymer, pressed and dried in place between the first and second layers.   30. The device of claim 28, wherein the first layer is an absorbent layer that absorbs the fluid sample and deploys on the device.   29. The apparatus of claim 28, wherein the first layer is an absorbent layer that includes a reagent for reacting with an analyte in the sample.   30. The apparatus of claim 28, wherein the first layer is an absorbent layer comprising a reagent for reacting with an interfering component of the sample.   30. The apparatus of claim 28, wherein the second layer is an absorbent layer that absorbs and retains components from the sample.   29. The apparatus of claim 28, wherein the second layer is an absorbent layer that includes a reagent for reacting with an analyte in the sample.   32. The apparatus of claim 31, wherein the component from the sample is a product of an analyte reaction in the first absorbent layer.   30. The apparatus of claim 28, wherein the liquid permeable adhesive layer is capable of performing physical separation of the fluid sample.   30. The apparatus of claim 28, wherein the liquid permeable adhesive layer is capable of reacting with a component of the sample, thereby trapping the component in the adhesive layer.   30. The apparatus of claim 28, wherein the liquid permeable adhesive layer contains an additive that can react with components of the sample, thereby preventing the components from passing through the adhesive layer.   29. The device of claim 28, wherein the water dispersible polymer is an anionic polyurethane dispersion combined with a water soluble polymer.   29. The device of claim 28, wherein the water dispersible polymer is an anionic polyurethane dispersion combined with a cationic acrylic dispersion as a water soluble polymer.   30. The device of claim 28, wherein the water dispersible polymer is a cationic polyurethane dispersion combined with a water soluble polymer.   41. The device of claim 38 or 40, wherein the water soluble polymer is at least one member of the group consisting of polyethylene oxide, polyvinyl pyrrolidone and polyvinyl alcohol.   30. The apparatus of claim 29, wherein the first absorbent layer is filter paper.   The group for the reaction with the analyte in the first absorbent layer is composed of oxidase, reductase and protease used in clinical trials, and antibody, nucleic acid, antigen and protein used in binding tests 32. The apparatus of claim 30, wherein the apparatus is an element of:   The reagent for reacting with the interference component of the sample comprises an enzyme for metabolizing the interference substance, a reactant for converting the interference substance into a non-reactive form, and a binder for capturing the interference substance 32. The apparatus of claim 31, wherein the apparatus is an element of:   The apparatus of claim 32, wherein the second absorbent layer is filter paper.   The reagent for reacting with the analyte in the second absorbent layer is a member of the group consisting of an indicator that emits a signal in response to the analyte and an enzyme or reactant for signal amplification; 30. The apparatus of claim 28.   The group of analyte reactions in the first layer consists of enzymes used in clinical trials and affinity binders used in binding tests and reactions in which a portion of the analyte is cleaved 35. The apparatus of claim 34, detected by an element of:   38. The additive to the adhesive layer capable of reacting with components of the sample is a member of the group consisting of an affinity binder or enzyme to remove interfering substances or emit a signal. Equipment.   Further comprising an additional absorbent layer disposed between the first and second absorbent layers, each of the additional absorbent layers being separated from the most adjacent absorbent layer by a further adhesive layer; 29. The device of claim 28, wherein the adhesive layer is diffusible into the fluid and comprises a blend of an aqueous polymer dispersion and a water soluble polymer that has been cast and dried to form the adhesive layer. .   29. A method for detecting an analyte in a fluid sample, the method comprising applying the sample to the multilayer device of claim 28 and measuring the amount of the analyte present in the sample. For detecting analytes in liquid samples,
(A) at least one reservoir for receiving the sample;
(B) at least one reservoir containing a dry reagent for receiving at least a portion of the sample and reacting with the analyte;
(C) a microfluidic device comprising the reservoir of (a) and (b) and a capillary passage through which liquid can travel;
A liquid permeable composition comprising a blend of an aqueous polymer dispersion and a water soluble polymer dried to form a liquid permeable composition, wherein the composition is disposed in at least one of the reservoirs. A microfluidic device that removes a component of the liquid sample or reacts with a component of the liquid sample.   52. The microfluidic device of claim 51, wherein the liquid permeable composition is an adhesive layer that separates a dry reagent or absorbent layer in a multilayer structure disposed in one of the reservoirs.   52. The microfluidic device of claim 51, wherein the liquid permeable composition is disposed at an entrance of the capillary passage to at least one of the reservoirs.   52. The microfluidic device of claim 51, wherein the liquid permeable composition is disposed at the outlet of a capillary passage to at least one of the reservoirs.   52. The microfluidic device of claim 51, wherein the liquid permeable composition includes an additive that chemically reacts with a component in the liquid sample.   56. The microfluidic device of claim 55, wherein the additive comprises an indicator dye or particle for producing a detectable response.   57. The microfluidic device of claim 56, wherein the additive comprises an exchange resin for removing buffer components and an ascorbate scavenger for eliminating ascorbate interference.   57. The microfluidic device of claim 56, wherein the additive comprises particles and a polymer for emitting a detectable response or removing an interfering component.   57. The microfluidic device of claim 56, wherein the additive comprises a metal and a chelating compound for producing a detectable response or removing an interfering component.   57. The microfluidic device of claim 56, wherein the additive comprises an enzyme for producing a detectable response or removing an interfering component.   57. The microfluidic device of claim 56, wherein the additive comprises an antibody or other affinity molecule for generating a detectable response or for separating interference components.   57. The microfluidic device of claim 56, wherein the additive comprises a filler for adjusting opacity or reflectivity.   57. The microfluidic device of claim 56, wherein the additive comprises a surfactant to increase fluid flow.   52. The microfluidic device of claim 51, wherein the liquid permeability of the liquid permeable composition is adjusted by changing the ratio of the aqueous polymer dispersion to the water soluble polymer.   52. The microfluidic device of claim 51, wherein the aqueous polymer dispersion is a polyurethane dispersion and the water soluble polymer is at least one member of the group consisting of polyethylene oxide, polyvinyl pyrrolidone and polyvinyl alcohol.   52. The microfluidic device of claim 51, wherein the ratio of the aqueous polymer dispersion to the water soluble polymer is 50: 1 to 1: 1 on a weight basis.

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